mouse anti human adam10 11g2  (Valiant Co Ltd)

Journal: eLife

Article Title: Proteomic landscape of tunneling nanotubes reveals CD9 and CD81 tetraspanins as key regulators

doi: 10.7554/eLife.99172

Figure Lengend Snippet:

Article Snippet: Antibody , Mouse monoclonal anti-ADAM10 11G2 , , Diaclone: #857.800.000 , WB (1/1000).

Techniques: Transfection, Construct, Expressing, Plasmid Preparation, Marker, Sequencing, Purification, Transduction, Control, Software, Staining

Journal: bioRxiv

Article Title: Tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1101/800557

Figure Lengend Snippet: (A) ADAM10-knockout MEFs (–) and ADAM10-knockout MEFs stably overexpressing FLAG-tagged Tspan15 (+) were lysed in 1% Triton X-100 lysis buffer and subjected to anti-FLAG (top panel) and anti-α-tubulin (bottom panel) western blotting. (B) Wild-type (WT) and Tspan15-knockout (KO) Jurkat human T cells were analysed by flow cytometry with tissue culture supernatant for each of the four mouse anti-human Tspan15 hybridomas (1C12, 4A4, 5D4 or 5F4; solid line), or with mouse IgG1 as a negative control (dotted line). Histograms are representative of two independent experiments. (C) HEK-293T cells were transfected with FLAG-tagged human TspanC8 expression constructs (except for Tspan10, which was of mouse origin) or an empty vector control (–), lysed in 1% Triton X-100 lysis buffer and western blotted with tissue culture supernatants for each of the four Tspan15 hybridomas, or a positive control FLAG antibody. Blots are representative of three independent experiments. (D) Human platelets were lysed in 1% digitonin lysis buffer and subjected to immunoprecipitation with the four Tspan15 mAbs or a negative control mouse IgG1, followed by anti-ADAM10 and anti-Tspan15 (5D4) western blotting (upper panels). The faint additional band in the 5F4 lane corresponds to light chain from the immunoprecipitating mAb (data not shown). To quantitate the data, the amount of ADAM10 co-immunoprecipitated was normalised to the amount of immunoprecipitated Tspan15 with each antibody (lower panel). Error bars represent standard error of the mean from three independent experiments.

Article Snippet: Primary antibodies were rabbit anti-FLAG (Sigma-Aldrich, Poole, UK), mouse or rabbit anti-α-tubulin (Cell Signaling Technology, London, UK), mouse anti-human ADAM10 11G2 ( Arduise et al. 2008 ), mouse anti-Tspan5 TS52 ( Saint-Pol et al. 2017 ) and negative control mouse IgG1 MOPC-21 (MP Biomedicals, Santa Ana, CA).

Techniques: Knock-Out, Stable Transfection, Lysis, Western Blot, Flow Cytometry, Negative Control, Transfection, Expressing, Construct, Plasmid Preparation, Control, Positive Control, Immunoprecipitation

Journal: bioRxiv

Article Title: Tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1101/800557

Figure Lengend Snippet: (A) Tspan15 surface expression in wildtype (WT), Tspan15-knockout (KO) and ADAM10 KO Jurkat, HEK-293T and A549 cell lines were analysed by flow cytometry with anti-Tspan15 mAb 1C12 or mouse IgG1 negative control antibody. Tspan15 surface expression is presented as the geometric mean fluorescence intensity of Tspan15 staining relative to the control staining. Error bars represent standard error of the mean from three independent experiments. Data were log-transformed and statistically analysed by a one-way ANOVA with Dunnett’s multiple comparisons test (* p <0.05, ** p <0.01, *** p <0.001 compared to WT). (B) HUVECs were transfected with two different ADAM10 siRNAs or a negative control siRNA and surface expression of ADAM10 and Tspan15 was measured by flow cytometry and analysed as described in panel A. (Ci) WT, Tspan15 KO and ADAM10 KO Jurkat cells were lysed in 1% Triton X-100 lysis buffer, immunoprecipitated with anti-Tspan15 mAb 5D4, and western blotted with the same antibody. Whole cell lysates were blotted with anti-α-tubulin mAb. (Cii) Tspan15 levels from panel Ci were quantitated and normalised to WT expression. Error bars represent standard errors of the mean from three independent experiments. Data were log-transformed and statistically analysed by a one-way ANOVA with a Tukey’s multiple comparisons test (*** p <0.001 compared to WT). (D) Tspan15 mRNA level in WT and ADAM10 KO Jurkat, HEK-293T and A549 cells were assessed by qRT-PCR and presented relative to GAPDH housekeeping gene expression. Error bars represent standard errors of the mean from three independent experiments. Data were log-transformed and statistically analysed by a two-way ANOVA followed by Tukey’s multiple comparisons test (n.s., not significant). (E) WT and ADAM10 KO HEK-293T cells were transfected with empty vector control (−) or Tspan15 (+) expression constructs. Tspan15 surface expression was measured by flow cytometry as described in panel A. Histograms are representative of four independent experiments.

Article Snippet: Primary antibodies were rabbit anti-FLAG (Sigma-Aldrich, Poole, UK), mouse or rabbit anti-α-tubulin (Cell Signaling Technology, London, UK), mouse anti-human ADAM10 11G2 ( Arduise et al. 2008 ), mouse anti-Tspan5 TS52 ( Saint-Pol et al. 2017 ) and negative control mouse IgG1 MOPC-21 (MP Biomedicals, Santa Ana, CA).

Techniques: Expressing, Knock-Out, Flow Cytometry, Negative Control, Fluorescence, Staining, Control, Transformation Assay, Transfection, Lysis, Immunoprecipitation, Western Blot, Quantitative RT-PCR, Gene Expression, Plasmid Preparation, Construct

Journal: bioRxiv

Article Title: Tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1101/800557

Figure Lengend Snippet: (A) ADAM10 surface expression in WT, ADAM10 KO and Tspan15 KO Jurkat, HEK-293T and A549 cells was measured by flow cytometry and quantitated as described in . (B) HUVECs were transfected with two different Tspan15 siRNAs or negative control siRNA and surface expression of ADAM10 was measured by flow cytometry and analysed as described in .

Article Snippet: Primary antibodies were rabbit anti-FLAG (Sigma-Aldrich, Poole, UK), mouse or rabbit anti-α-tubulin (Cell Signaling Technology, London, UK), mouse anti-human ADAM10 11G2 ( Arduise et al. 2008 ), mouse anti-Tspan5 TS52 ( Saint-Pol et al. 2017 ) and negative control mouse IgG1 MOPC-21 (MP Biomedicals, Santa Ana, CA).

Techniques: Expressing, Flow Cytometry, Transfection, Negative Control

Journal: bioRxiv

Article Title: Tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1101/800557

Figure Lengend Snippet: (A) Schematic representation of ADAM10 tagged with the C-terminal half of superfolder GFP (sfGFP-C), Tspan15 tagged with the N-terminal half of superfolder GFP (sfGFP-N) and the predicted ADAM10/Tspan15 BiFC dimer. Solid ovals represent N-glycosylation. (B) HEK-293T cells were transfected with the ADAM10 and Tspan15 BiFC expression constructs, fixed and stained with Alexa Fluor® 647-conjugated Tspan15 mAb 5D4, and analysed by confocal microscopy. The image shown is representative of middle plane sections taken from two independent experiments (scale bar 10 µm). (C-D) Fluorescence correlation spectroscopy (FCS) measurements from the upper membrane of HEK-293T expressing the ADAM10/Tspan15 BiFC complexes were used to determine the average particle concentration (C) and diffusion co-efficient (D) of the complexes. (E) Fluorescence fluctuations from the FCS reads were also subjected to photon counting histogram (PCH) analysis to obtain the average molecular brightness (ε) of particles within the confocal volume. The FCS data were separated into groups that preferentially fit to a one-component or a two-component PCH model with dimmer and brighter subcomponents. Data were obtained from 43 individual measurements from three independent experiments. Error bars represent standard errors of the mean, N is the number of particles, and cpm is the counts per molecule. Data were log-transformed and statistically analysed by a one-way ANOVA followed by Tukey’s multiple comparisons test (*** p <0.001).

Article Snippet: Primary antibodies were rabbit anti-FLAG (Sigma-Aldrich, Poole, UK), mouse or rabbit anti-α-tubulin (Cell Signaling Technology, London, UK), mouse anti-human ADAM10 11G2 ( Arduise et al. 2008 ), mouse anti-Tspan5 TS52 ( Saint-Pol et al. 2017 ) and negative control mouse IgG1 MOPC-21 (MP Biomedicals, Santa Ana, CA).

Techniques: Glycoproteomics, Transfection, Expressing, Construct, Staining, Confocal Microscopy, Fluorescence, Spectroscopy, Membrane, Concentration Assay, Diffusion-based Assay, Transformation Assay

Journal: bioRxiv

Article Title: Tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1101/800557

Figure Lengend Snippet: (A) Schematic representation of the synthetic ADAM10/Tspan15 fusion protein that has the C-terminus of ADAM10 physically linked to the N-terminus of Tspan15. Solid ovals represent N-glycosylation. (B) ADAM10/Tspan15 double KO HEK-293T cells were transfected with ADAM10/Tspan15 fusion construct, lysed in 1% digitonin lysis buffer in the presence of 10 µM ADAM10 inhibitor GI254023X, to prevent post-lysis auto-proteolysis, and western blotted for ADAM10 and Tspan15. (C) Cells described in panel B were assessed for surface expression of Tspan15 (green) and ADAM10 (red) by flow cytometry. Black traces represent isotype control staining. (D) ADAM10/Tspan15 double KO HEK-293T cells were transfected with the indicated expression constructs and analysed by confocal microscopy using anti-ADAM10 (green) and Tspan15 (red) mAbs. The cells were non-permeabilised and images are maximum intensity projections of confocal z-stacks. The scale bar on the upper left image is 30 µm. (Ei) ADAM10/Tspan15 double KO HEK-293T cells were co-transfected with alkaline phosphatase-tagged betacellulin (BTC) and Tspan15, ADAM10, ADAM10 and Tspan15, the ADAM10/Tspan15 fusion, or an empty vector control. Cells were stimulated with 2 mM NEM or vehicle control and alkaline phosphatase activity was measured in the supernatant and whole cell lysates to quantitate the percentage of BTC shed. Data were arcsine-transformed and statistically analysed with a two-way ANOVA followed by a Tukey’s multiple comparisons test (*** p <0.001 compared to empty vector-transfected control). (Eii) Transfected cells were assessed for surface expression of ADAM10 by flow cytometry and the data quantitated and analysed using a paired t-test (* p <0.05).

Article Snippet: Primary antibodies were rabbit anti-FLAG (Sigma-Aldrich, Poole, UK), mouse or rabbit anti-α-tubulin (Cell Signaling Technology, London, UK), mouse anti-human ADAM10 11G2 ( Arduise et al. 2008 ), mouse anti-Tspan5 TS52 ( Saint-Pol et al. 2017 ) and negative control mouse IgG1 MOPC-21 (MP Biomedicals, Santa Ana, CA).

Techniques: Glycoproteomics, Transfection, Construct, Lysis, Western Blot, Expressing, Flow Cytometry, Control, Staining, Confocal Microscopy, Plasmid Preparation, Activity Assay, Transformation Assay